U.S. patent number 6,900,055 [Application Number 09/830,592] was granted by the patent office on 2005-05-31 for preparation of porous silicone rubber for growing cells or living tissue.
This patent grant is currently assigned to Cellon S.A.. Invention is credited to Robert McLean Bird, Tony Clayson, Timothy Burgess Clifford, Jess Paul Fuller, David Pegg.
United States Patent |
6,900,055 |
Fuller , et al. |
May 31, 2005 |
Preparation of porous silicone rubber for growing cells or living
tissue
Abstract
A method of making a silicone rubber having a structure adapted
for growth of cells or living tissue, which comprises contacting a
silicone rubber precursor with a biologically-acceptable
sacrificial filler, curing the resultant mixture and removing the
sacrificial filler to form a structured silicone rubber. The
sacrificial filler is preferably an inorganic salt that has been
ground, and the salt is selected from metal halides, metal
carbonates and metal bicarbonates.
Inventors: |
Fuller; Jess Paul
(Leicestershire, GB), Pegg; David (York,
GB), Bird; Robert McLean (Derby Shire, GB),
Clifford; Timothy Burgess (Leicestershire, GB),
Clayson; Tony (Leicestershire, GB) |
Assignee: |
Cellon S.A. (Bereldange,
LU)
|
Family
ID: |
26314572 |
Appl.
No.: |
09/830,592 |
Filed: |
August 13, 2001 |
PCT
Filed: |
October 28, 1999 |
PCT No.: |
PCT/GB99/03558 |
371(c)(1),(2),(4) Date: |
August 13, 2001 |
PCT
Pub. No.: |
WO00/24437 |
PCT
Pub. Date: |
May 04, 2000 |
Foreign Application Priority Data
|
|
|
|
|
May 28, 1998 [GB] |
|
|
9912641 |
Oct 28, 1998 [GB] |
|
|
9823446 |
|
Current U.S.
Class: |
435/395; 424/423;
424/93.7; 435/180; 435/182 |
Current CPC
Class: |
A61P
5/00 (20180101); A61L 29/06 (20130101); A61L
15/26 (20130101); C12M 23/24 (20130101); A61P
3/10 (20180101); A61P 31/00 (20180101); C12M
25/02 (20130101); A61L 27/18 (20130101); A61P
43/00 (20180101); C12M 21/08 (20130101); C08J
9/26 (20130101); A61L 15/26 (20130101); C08L
83/04 (20130101); A61L 27/18 (20130101); C08L
83/04 (20130101); A61L 29/06 (20130101); C08L
83/04 (20130101); C08J 2383/04 (20130101); C08J
2201/0442 (20130101) |
Current International
Class: |
A61L
15/26 (20060101); A61L 15/16 (20060101); A61L
29/00 (20060101); A61L 29/06 (20060101); A61L
27/00 (20060101); A61L 27/18 (20060101); C08J
9/26 (20060101); C08J 9/00 (20060101); C12M
3/06 (20060101); A61F 002/00 (); C12N 005/06 ();
C12N 005/08 (); C12N 011/08 (); C12N 011/04 () |
Field of
Search: |
;435/177,180,182,395
;424/423,93.7 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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|
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|
-0 277 009 |
|
Aug 1988 |
|
EP |
|
-0 326 278 |
|
Aug 1989 |
|
EP |
|
-0 450 671 |
|
Oct 1991 |
|
EP |
|
WO 94/16058 |
|
Jul 1994 |
|
WO |
|
WO 96/20050 |
|
Jul 1996 |
|
WO |
|
WO-97/03744 |
|
Feb 1997 |
|
WO |
|
WO 97/08291 |
|
Mar 1997 |
|
WO |
|
WO-97/15242 |
|
May 1997 |
|
WO |
|
WO-97/21347 |
|
Jun 1997 |
|
WO |
|
Primary Examiner: Naff; David M.
Attorney, Agent or Firm: Ropes & Gray, LLP
Claims
What is claimed is:
1. A porous silicone rubber article having a structure adapted for
growth of cells or living tissue obtained by a method comprising
mixing a biologically acceptable sacrificial filler with a silicone
rubber precursor, curing the resultant mixture at a temperature
below 180.degree. C., and removing the sacrificial filler by
dissolution to form a porous silicone rubber article, said
sacrificial filler is an inorganic salt that has been ground, and
said inorganic salt is selected from the group consisting of metal
halides, metal carbonates and metal bicarbonates.
2. A biomedical device comprising a porous silicone rubber article
as claimed in claim 1.
3. A method of making a silicone rubber article having a structure
adapted for growth of cells or living tissue, which comprises
mixing a biologically acceptable sacrificial filler with a silicone
rubber precursor, curing the resultant mixture at a temperature
below 180.degree. C., and removing the sacrificial filler by
dissolution to form a porous silicone rubber, said sacrificial
filler is an inorganic salt that has been ground, and said
inorganic salt is selected from the group consisting of metal
halides, metal carbonates and metal bicarbonates.
4. A method as claimed in claim 3, wherein the silicone rubber
precursor can be cured or vulcanized at room temperature.
5. A method as claimed in claim 3 or 4, wherein the
biologically-acceptable sacrificial filler is bio-compatible, such
that it is innately non-toxic and does not leave a toxic
residue.
6. A method as claimed in claim 3 or 4, wherein the sacrificial
filler does not interact chemically with the silicone rubber
precursor or with the resultant silicone rubber and is stable at
temperatures used to cure the resultant mixture.
7. A method as claimed in claim 3 or 4, wherein the sacrificial
filler is granular.
8. A method as claimed in claim 3 or 4, wherein the sacrificial
filler is amorphous.
9. A method as claimed in claim 3, wherein the sacrificial filler
is milled to a particle size of 0.01-10 .mu.m.
10. A method as claimed in claim 3, wherein the sacrificial filler
is milled in an organic solvent.
11. A method as claimed in claim 3, wherein the inorganic salt is
selected from the group consisting of lithium bicarbonate, sodium
bicarbonate, potassium bicarbonate, lithium chloride, sodium
chloride and potassium chloride.
12. A method as claimed in claim 11, wherein the sacrificial filler
is sodium bicarbonate or sodium chloride.
13. A method as claimed in claim 12, wherein the sodium bicarbonate
or sodium chloride is wet-milled under xylene.
14. A method as claimed in claim 3, wherein the sacrificial filler
is removed by dissolution.
15. A method as claimed in claim 3, wherein the sacrificial filler
does not cause swelling of the silicone rubber when removed using
an aqueous solvent.
16. A method as claimed in claim 15, wherein the sacrificial filler
is sodium bicarbonate.
17. A method as claimed in claim 3, wherein free --OH groups of the
silicone rubber are chemically modified, so as to enhance cell
adherence.
18. A method as claimed in claim 3, wherein the surface of the
silicone rubber is charged by bombardment with electrons.
19. A method as claimed in claim 3, wherein the silicone rubber
precursor comprises at least one additive that is not removed with
the sacrificial filler and serves to impart desired physical
properties to the rubber.
20. A method as claimed in claim 19, wherein the additive is a
metal powder or carbon black and serves to render the silicone
rubber electrically conductive.
21. A method as claimed in claim 20, wherein the additive is
stainless steel powder.
22. A method as claimed in claim 20, wherein the additive is iron
oxide.
23. A method as claimed in claim 19, wherein the additive is an
inert substance, and serves to render the silicone rubber
mechanically rigid.
24. A method as claimed in claim 3, wherein a surface of the
silicone rubber precursor is contacted with the sacrificial filler,
so as to form a structured silicone rubber having a textured
surface.
25. A method as claimed in claim 24, wherein the textured surface
of the silicone rubber facilitates attachment of adherent
cells.
26. A method as claimed in claim 24 or 25, wherein the textured
surface of the silicone rubber provides an increased number of
sites for attachment of cells relative to an untextured
surface.
27. A method as claimed in claim 3, wherein pores of the silicone
rubber provide sites of attachment for cells.
28. A method as claimed in claim 3, wherein the resultant mixture
is shaped prior to curing.
29. A method as claimed in claim 3, wherein pores of the silicone
rubber are 1 .mu.m-0.5 mm in diameter.
30. A method as claimed in claim 3, wherein the porous silicone
rubber is cut to a desired size or shape.
31. A method as claimed in claim 5 wherein the sacrificial filler
is crystalline.
32. A method as claimed in claim 3 wherein the sacrificial filler
is classified prior to contacting the silicone rubber
precursor.
33. A method as claimed in claim 9, wherein the sacrificial filler
is milled to a particle size of 0.05-1 .mu.m.
34. A method as claimed in claim 9, wherein the sacrificial filler
is milled to a particle size of 0.1-0.4 .mu.m.
35. A method as claimed in claim 3, wherein the sacrificial filler
is removed by dissolution in an aqueous solvent.
36. A method as claimed in claim 23, wherein the additive is glass,
and serves to render the silicone rubber mechanically rigid.
37. A method as claimed in claim 28, wherein the resultant mixture
is shaped prior to curing, by molding or extrusion.
38. A method as claimed in claim 29, wherein the pores are 10
.mu.m-0.2 mm in diameter.
39. A method as claimed in claim 29, wherein the pores are 50 to
150 .mu.m in diameter.
Description
BACKGROUND OF THE INVENTION
The present invention relates to methods for manufacturing silicone
rubber that is adapted to promote cell adhesion and growth, and, in
particular, to methods for providing silicone rubber with a
modified surface or structure for enhanced cell attachment. The
resultant silicone rubber is well-suited to a variety of tissue
culture and medical applications.
Silicones surpass other elastomers in many performance categories
because of their rigid silicon-oxygen chemical structure. The
process of vulcanisation transforms this structure, allowing the
silicon-oxygen polymer to become an elastic rubber. Silicone
rubbers are stable throughout a temperature range of -46.degree. C.
to 232.degree. C. They are odourless, tasteless and do not support
bacterial growth. Silicone rubbers also do not stain or corrode
with other materials. Most importantly, silicone rubbers are not
physically or chemically degraded or altered by contact with body
fluids, are not toxic or allergenic to human tissue and will not
excite an inflammatory or foreign body reaction. Silicone rubbers
can be formulated and tested for full bio-compatibility and
compliance with guidelines for medical products. A further and
particularly important advantage of silicone rubbers is that they
have the highest oxygen permeability of known polymers.
Forming textured and porous silicone rubber allows all of these
advantageous properties of silicone rubber to be exploited and
enhanced. For example, a textured surface will not only greatly
increase the available surface area for cell attachment, but will
also encourage cell attachment. Furthermore, the increased surface
area will increase the oxygen permeating through the silicone,
enhancing the metabolic activity of the cells attached thereto.
These advantages are very important in the various applications of
textured and porous silicone rubbers discussed below.
SUMMARY OF THE INVENTION
In a first aspect of the invention, there is provided a method of
making a silicone rubber having a structure adapted for growth of
cells or living tissue, which comprises contacting a silicone
rubber precursor with a biologically-acceptable sacrificial filler,
curing the resultant mixture and removing the sacrificial filler to
form a structured silicone rubber. Any suitable silicone rubber
precursor may be used, depending upon the intended application of
the resultant structured silicone rubber. Such silicone rubber
precursors are widely available commercially, for example, from Dow
Chemical Corporation, Midland, Mich., USA, or from GE Silicones
Europe, Bergen op Zoom, the Netherlands. In a preferred embodiment,
the silicone rubber precursor is one that can be cured or
vulcanised at room temperature. This obviates the need to expose
the mixture to elevated temperatures, which is particularly useful
as some sacrificial fillers become unstable and decompose at
elevated temperatures, thus making it difficult to control the
final form of the structured silicone rubber. In a further
embodiment, the biologically-acceptable sacrificial filler is
biocompatible, such that it is innately non-toxic and does not
leave a toxic residue. This is of particular importance where the
structured silicone rubber is intended for use in tissue culture
and medical applications, although a number of further factors also
need to be considered when choosing a suitable sacrificial filler.
For example, the sacrificial filler should preferably not react
with the silicone rubber, either in its precursor form or in its
cured state. The filler should also preferably be soluble in order
to facilitate its removal by dissolution and the solvent used to
dissolve the material should preferably not react with the silicone
rubber. If the silicone rubber is to be cured at elevated
temperatures, it is usually desirable to use a sacrificial filler
that is stable at the curing temperatures, since materials that
melt or decompose at high temperatures may be unsuitable,
particularly if a structured silicone rubber having a high degree
of regularity is desired. Finally, for commercial reasons, it is
generally desirable that the sacrificial filler should be
relatively inexpensive and readily available. In a preferred
embodiment, the sacrificial filler is ground, prior to contacting
the silicone rubber precursor. This has the advantage of allowing
the resultant structure of the silicone rubber to be controlled
much more accurately. Any suitable method for grinding the
sacrificial filler may be used, although it has been found that
wet-milling the sacrificial filler, prior to mixing with the
silicone rubber precursor, gives good results. However, the
sacrificial filler may also be ground by dry milling, preferably
under an inert or dry atmosphere, such as under dry nitrogen or
argon gas. In a preferred embodiment, the sacrificial filler is
milled to a particle size of 0.01-10 .mu.m, preferably 0.05-1
.mu.m, and most preferably 0.1-0.4 .mu.m. In a further embodiment,
the sacrificial filler is granular and, preferably, crystalline,
although certain amorphous fillers may also be suitable. Inorganic
salts have been found to give particularly good results, although
certain crystalline organic compounds, such as simple saccharides,
may often be equally effective. Where the sacrificial filler is an
inorganic salt, it is especially preferred to grind it first by
milling it in an organic solvent, since this gives good control
over resultant particle size. Preferably, the sacrificial filler is
an inorganic salt selected from the group consisting of metal
halides, metal carbonates and metal bicarbonates, especially one
selected from the group consisting of lithium bicarbonate, sodium
bicarbonate, potassium bicarbonate, lithium chloride, sodium
chloride and potassium chloride. In an especially preferred
embodiment, the sacrificial filler is sodium bicarbonate or sodium
chloride, preferably of high purity, such as food grade sodium
bicarbonate or sodium chloride. In this last embodiment, the sodium
bicarbonate or sodium chloride is preferably wet-milled under
xylene, although other volatile organic solvents may also be used.
In a further embodiment, the ground sacrificial filler is
classified, prior to contacting the silicone rubber precursor to
ensure uniform particle size distribution, for example, by passing
the ground material through sieves or by using a Malvern.RTM.
Particle Sizer. In another embodiment, the sacrificial filler is
removed by dissolution, preferably in an aqueous solvent. In the
latter case, the sacrificial filler is desirably chosen so that it
does not cause swelling of the silicone rubber when removed with an
aqueous solvent. In a further embodiment, at least a portion of the
free --OH groups that are normally present in the silicone rubber
are chemically modified, so as to enhance or promote cell
adherence. For example, free --OH groups may be chemically
converted to form positively charged groups, for example, by
reaction with diethylaminoethylbromide to give DEAE moieties, or to
form negatively charged groups, for example, by reaction with
iodoacetic acid, to give carboxylate moieties. In an alternative
embodiment, the surface of the silicone rubber may be charged
electrostatically, for example, by bombardment with electrons.
Alternatively, the surface characteristics of the silicone rubber
may be modified by applying a thin coating of a suitable polymer,
so as to make it more adherent to certain cells, whilst still
retaining a sufficient degree of gas permeability. Any suitable
polymer may be used, such as one selected from the group consisting
of polyolefins, polyvinyl resins, polyester resins, polyurethanes,
polyamines, polyamides, polyethers and polysaccharides. In a
preferred embodiment, the silicone rubber precursor also includes
at least one additive that is not removed with the sacrificial
filler and serves to impart desired physical properties on the
resultant silicone rubber. For example, the additive may be a metal
powder or carbon black, which can be used to render the silicone
rubber electrically conductive. Alternatively, the additive may be
stainless steel powder or iron oxide, which can be used to increase
the density of the silicone rubber. The additive may also be an
inert substance, such as glass, which can be used to render the
silicone rubber mechanically rigid. However, many other suitable
additives will also be apparent to those skilled in the art.
In a second aspect of the invention, there is provided a method of
making a silicone rubber having a structure adapted for growth of
cells or living tissue substantially in accordance with the
invention in its first aspect, wherein a surface of the silicone
rubber precursor is contacted with the sacrificial filler, so as to
form a structured silicone rubber having a textured surface. The
textured surface of the silicone rubber helps to facilitate
attachment of adherent cells, as well as providing an increased
surface area and, thus, number of sites for attachment of cells
relative to an untextured surface. In an embodiment, the inventive
method comprises forming a coating of a silicone rubber precursor
on a substrate, contacting a surface of the coating with a
biologically-acceptable sacrificial filler, curing the resultant
mixture and removing the sacrificial filler to form a textured
silicone rubber. Suitable silicone rubber precursors and
biologically acceptable sacrificial fillers are essentially as
described above in relation to the invention in its first aspect.
In a preferred embodiment, the surface of the coating is contacted
with the sacrificial filler under pressure, such that the
sacrificial filler is substantially completely embedded in the
coating. For example, the sacrificial filler may be dry-sprayed on
to the surface of the coating, or may be applied loosely to the
surface of the coating and then embedded by contacting the surface
with a pressure roller. Preferably, the sacrificial filler is
embedded to a depth of 0.1-1.0 mm, more preferably 0.1-0.5 mm, and
more preferably 0.1-0.25 mm. In an alternative embodiment, the
sacrificial filler is scattered or sprinkled over the surface of
the coating, such that the sacrificial filler is only partially
embedded in the surface. The latter technique can be used to
provide the surface of the silicone rubber with a less uniform
texture that is particularly suitable for growing certain types of
adherent cells. Preferably, the resultant textured surface is
micro-cupulated, i.e., cratered or pitted, the micro-cupules having
a depth of less than 1 mm, preferably a depth of 0.5-0.1 mm. In a
preferred embodiment, the micro-cupules measure less than 2 mm
across, preferably less than 1 mm across, and, most preferably,
less than 0.5 mm across. Silicone rubbers are available with a wide
range of different physical properties, both in the uncured and
cured state, and their methods of cure also differ widely.
Consequently, the nature and properties of the silicone rubber used
can affect the manufacturing process and the choice of a suitable
silicone rubber precursor can be important. The silicone rubber
precursor should be selected with due consideration to the manner
in which the mixture is to be applied to the substrate, the
conditions required for curing, and the desired properties of the
end product. The uncured silicone rubber should usually have an
appropriate viscosity for the method of its application to the
substrate, and should retain its general form once the sacrificial
filler has adhered to its surface. The conditions for curing must
generally be compatible with both the substrate to which the
uncured silicone rubber is applied and the sacrificial filler that
adheres to the surface. Finally, the quality of the silicone rubber
used should be selected in light of the intended application of the
final product. In an especially preferred embodiment, silicone
rubber paint RTV 118 (General Electric Co., Connecticut, USA) is
used. In order to assist adhesion of the silicone rubber layer to
certain materials, it may be necessary to apply a conventional
adhesive, such as a mineral spirit-based primer, prior to
deposition of the silicone layer. In a preferred embodiment, the
primer used is silicone rubber primer SS 4155 (General Electric
Co., Connecticut, USA). The micro-cupulated silicone rubber
surfaces formed by the inventive method may be formed on or applied
to any suitable substrates. When applied to cell culture vessels,
such as culture flasks or roller bottles, the textured silicone
rubber surfaces have been found to produce greatly increased yields
in tissue culture processes. Such surfaces provide increased
surface area for cell attachment, as well as promoting or
encouraging cell attachment. The increased surface area also
enhances oxygen supply to the surface. Thus, textured silicone
layers according to the invention may be used in a variety of
devices, particularly those where cell attachment is important.
In a third aspect of the invention, there is provided a method of
making a silicone rubber having a structure adapted for growth of
cells or living tissue substantially in accordance with the
invention in its first aspect, wherein the sacrificial filler is
dispersed throughout the silicone rubber precursor, and the
structured silicone rubber is substantially porous. In this aspect,
the inventive method creates a system of pores and channels
throughout the silicone rubber structure. The pores of the silicone
rubber provide sites of attachment for cells or tissues, so that
the cells or tissues may be substantially trapped within the
resultant structure. This system of pores can also act as a
capillary system, increasing oxygen and nutrient supply to the
surface of the structure. In a preferred embodiment, the method of
making a porous silicone rubber comprises mixing the
biologically-acceptable sacrificial filler with the silicone rubber
precursor, curing the resultant mixture at a temperature below
180.degree. C., and removing the sacrificial filler, to form a
porous silicone rubber. Preferably, the silicone rubber is cured at
a temperature between 100.degree. C. and 175.degree. C., more
preferably between 120.degree. C. and 170.degree. C., more
preferably between 140.degree. C. and 160.degree. C., and most
preferably about 150.degree. C. The silicone rubber precursors and
the biologically acceptable sacrificial fillers that can be used
are essentially the same as described above in relation to the
invention in its first or second aspects. In another embodiment,
the resultant mixture may be shaped prior to curing, preferably by
moulding or extrusion. In a preferred embodiment, the average size
of the pores formed is 1 .mu.m-0.5 mm, preferably 10 .mu.m to 0.2
mm, and more preferably 50 to 150 .mu.m in diameter. Preferably,
the porous silicone rubber is cut to a desired size or shape. For
example, the porous silicone rubber may be cut in the form of small
pellets that are capable of allowing cell growth within their pores
but which can be readily separated from the culture medium by
traditional separation methods, such as centrifugation or
filtration. Silicone rubbers frequently contain innate fillers,
such as fumed glass, which are added to produce desired viscosity,
strength and other physical properties. The amount of sacrificial
filler that can be mixed into the silicone rubber and, therefore,
the extent of the porosity achieved is inversely proportional to
the quantity of innate filler already present. Thus, a low
viscosity silicone rubber containing small amounts of innate filler
can accommodate a greater packing density of sacrificial filler
than a high viscosity silicone rubber containing high levels of
innate filler to give it a thicker consistency. The viscosity of
the silicone rubber is also of importance when considering the
manner in which the mixture is to be manipulated to give the end
product. For example, if the mixture is to be extruded, a low
viscosity silicone rubber, although able to hold a greater amount
of sacrificial filler, may not be suitable, because separation of
the filler can occur, particularly if extrusions of small
cross-sections are required, as well as slumping of the mixture,
due to its low viscosity, which can result in distorted shapes.
However, if the mixture is to be spread into a sheet or moulded,
then a low viscosity silicone rubber may well be appropriate
because, for the same amount of filler, it is more easily
manipulated. The green strength, i.e., the strength of the uncured
silicone rubber precursor mixture, is also a factor for
consideration. Low viscosity silicone rubber, when packed with
sacrificial filler, for example, exhibits very poor green strength
and is, thus, generally undesirable for extrusion. For extrusion
applications, a very high viscosity silicone rubber would be ideal
in principle, but, as so little sacrificial filler can be mixed
into these materials, they are not usually a practical option.
Therefore, a silicone rubber somewhere between the two must be
chosen, such that sufficient innate filler can be included to
maintain green strength but insufficient to be able to pack in
sufficient sacrificial filler. The cure regime of the silicone
rubber must also be taken into account. Where a rapid cure is
required, for example, so as to maintain the geometry of an
extrusion, heat cure systems are often required. However, these
systems must be tailored such that the heat process does not have a
deleterious effect on the filler. It may also be necessary to use
room temperature curing systems if the material needs to be bound
to an additional substrate that is unable to withstand elevated
temperature, such as a thermoplastic, for example, The physical
properties of the cured silicone rubber must also be considered.
Where durability is an important issue, such as in the formation of
tubes or sheets, then a silicone rubber having high tensile
strength must be used. However, such silicone rubbers tend to be of
higher viscosity and contain large amounts of innate filler and,
hence, a compromise must be found. Where tensile strength is less
of an issue, a low viscosity silicone rubber may be used,
especially if there is no requirement for extrusion. Finally, the
actual grade of silicone rubber is worthy of note. The final
application of the material will determine the quality of the
silicone rubber to be used. For medical and implantable
applications, a high purity grade of material should be used, and,
conversely, industrial grade silicone rubbers may be appropriate
for applications, such as waste water treatment. In some instances,
it may be desirable to include additives in the mixture in order to
achieve certain characteristics, such as desired density, magnetic
properties and the like. In the majority of cases, such additives
would generally be in powder form and the considerations needed to
choose suitable materials would be similar to those for the
sacrificial filler. For example, if the silicone rubber is required
to have an increased density, a high mass powder would be added in
small quantities to make these adjustments and the choice of powder
would follow criteria such as reactivity, toxicity and economics,
etc. It has been found by the present invention that the use of
certain sacrificial fillers can have an adverse effect on the
resultant silicone rubber. For example, the use of sodium chloride
can cause the silicone rubber to swell, depending upon the
conditions. In order to avoid, this, it is desirable to use a
sacrificial filler that does not cause swelling or adversely effect
the resultant silicone rubber. Sodium bicarbonate has been found to
be particularly effective in satisfying such criteria, although a
number of other sacrificial fillers may be equally effective. If
sodium bicarbonate is to be used as a sacrificial filler, it
decomposes and, therefore, "blows" the material at temperatures
above approximately 180.degree. C. Consequently, it is necessary to
adapt the manufacturing process so as to avoid temperatures above
180.degree. C., for example, by selecting silicone rubbers, which
cure at lower temperatures. Many of the alternative sacrificial
fillers are toxic, leave toxic residues when dissolved, or are
problematic at moderate temperatures required for working with
silicone rubber. The silicone rubbers formed using the methods in
accordance with the first, second and third aspects of the
invention have properties that make them particularly well-suited
for use in a range of biomedical devices and apparatus.
In a fourth aspect of the invention, there is provided a culture
chamber for use in a method of culturing microbiological material,
which comprises at least one gas-permeable wall or portion of a
wall, and a textured interior growth surface arranged for contact
with the microbiological material being cultured. The general
principles of culturing cells in vitro are well-established in the
field of biotechnology, with the term "cell culture" being usually
understood to refer to both growth and maintenance of cells. In a
preferred embodiment, the gas-permeable wall and the textured
interior growth surface are each formed from an organic polymer,
optionally the same organic polymer. The gas-permeable wall or
potion of a wall of the culture chamber may also provide the
textured interior growth surface, such that cells may grow directly
on a textured growth surface on the gas-permeable membrane, thus
allowing high cell densities. In a preferred embodiment, the
gas-permeable wall or portion of a wall comprises a silicone rubber
membrane. In an especially preferred embodiment, the textured
interior growth surface is obtained or obtainable by a method
according to the invention in its second aspect. Preferably, the
culture chamber has at least one port extending between the
interior and the exterior of the chamber. More often, however,
there will be at least two ports, preferably including an inlet and
an outlet port. An additional septum port may also be provided, to
reduce the risk of contamination when introducing various
substances to the culture chamber. In an embodiment, at least one
or both of the inlet and outlet ports are septum ports. In an
especially preferred embodiment, the culture chamber is in the form
of a flexible bag or envelope. A variety of different apparatus is
known for culture of cells in vitro. In recent years, flexible
culture bags have become increasingly popular, offering a number of
advantages over traditional cell culture apparatus, such as
multi-well plates, flasks, roller bottles and spinner flasks. For
example, culture bags represent closed systems, thus reducing the
risk of contamination, as well as taking up less storage and
incubator space. In addition such culture bags can often be
produced relatively inexpensively, making them effectively
disposable and reducing any need to sterilise them for re-use. In
most tissue culture applications, aeration of the culture is
essential in order to provide the cells with oxygen necessary for
growth. In the past, methods such as sparging, surface aeration and
medium perfusion have been used to increase oxygen availability.
However, such methods can cause cellular damage, thereby severely
limiting the efficiency of cell culture. Silicone rubbers have the
highest oxygen permeability of known polymers, and tubing or
membranes made from such materials are well-suited for use in cell
culture, where they are able to provide improved diffusion of
oxygen to the cells. Silicone rubbers not only provides gas
permeability (including oxygen and carbon dioxide) but also vapour
transmission, structural integrity, resilience and temperature
resistance, all of which are desirable in cell and tissue culture.
International patent application no. PCT/US96/20050 (Avecor
Cardiovascular, Inc.) discloses a cell culture bag formed from a
plurality of thin, spaced, gas-permeable silicone membranes, whose
gas exchange rate is claimed to be significantly higher than most
conventional culture bags. However, although such bags may be
capable of sustaining higher cell densities and cell viability,
they are ultimately limited by the surface area of the bag.
Moreover, the interior surfaces of such bags are smooth and, thus,
provide poor cell attachment features, making them unsuitable for
efficient cell culture of anchorage-dependent cells. Furthermore,
certain "problem" cell types are unable to attach to the smooth
interior surface of the bags. An elaborate (and seemingly
expensive) method of increasing the surface area available for cell
adhesion is described in U.S. Pat. No. 4,937,194 (Baxter
International, Inc.), which discloses a flexible bag containing an
internal cellular structure, such as a honeycomb type structure
with hexagonal channels passing through it, serving as adherent
sites for cells being cultured. This document also proposes the use
of microcarriers, such as small glass spheres or sodium alginate,
to increase the surface area available for cell adherence inside
the bag. There is a need, therefore, to overcome some of the
aforementioned disadvantages. Accordingly, in an especially
preferred embodiment of the invention in its fourth aspect, there
is provided a culture chamber in the form of flexible bag or
envelope. Such a culture bag provides an increased growth substrate
surface area for cell attachment, as well as providing a growth
substrate that will assist cell attachment. Moreover, the bag
structure is simple and inexpensive to manufacture. In a preferred
embodiment, the bag is made from at least one silicone rubber sheet
that is coated with a silicone rubber layer having a rough or
uneven micro-cupulated growth surface exhibiting a plurality of
craters or crater-like depressions. Preferably, a room-temperature
vulcanising silicone rubber precursor is used, whilst the
sacrificial filler used to produce the textured surface is
preferably sodium chloride. The textured or micro-cupulated surface
so formed significantly increases the surface area for cell
attachment, thereby increasing the efficiency of cell culture. The
micro-cupulated surface also assists attachment and growth of
certain "problem" cell types, such as, for instance, stromal cells
necessary for stem cell expansion processes. Stromal cells
originating from bone require a textured surface on which to grow
if their proliferation is to be optimised. As already described
above in relation to the culture chamber, the culture bag
preferably also includes one or more ports, extending between the
bag interior and bag exterior. Such ports may be used for
introducing nutrient medium, taking samples, adding further
ingredients, etc. The ports should preferably have valves, locks or
the like, to avoid contamination of the big interior. In a
preferred embodiment, the culture bag is provided with an inlet and
an outlet port with luer locks, and a septum port for taking
samples or introducing substances into the bag. The ports are
desirably positioned between the sealed edges of the culture bag.
It has been found that the application of a textured surface to a
culture bag wall in accordance with the invention can result in the
wall becoming opaque. In a preferred embodiment, therefore, the
culture bag also includes at least one portion of membrane to which
no textured surface layer has been applied, this area serving to
act as a transparent window, thus allowing a user to see inside the
culture chamber. In a further embodiment, the culture chamber also
includes a valve means, allowing the release of gases that build up
during cell growth and may form an air bubble inside the bag. The
presence of a bubble within the chamber can prevent colonisation on
the surface area adjacent the bubble because the surface will not
be in contact with the culture medium. Thus, the presence of a
valve in the culture chamber wall helps to minimise the size of any
gas bubbles, thereby allowing a larger surface area of the bag to
remain in contact with the nutrient medium and to be available for
cell attachment. Almost complete colonisation on the interior
chamber surface is, therefore, possible, increasing the efficiency
of the culture chamber. Desirably, the valve comprises a filter
means, allowing gases to diffuse out of the chamber but preventing
microbial contamination. In a preferred embodiment, the valve means
comprises one or more layers of a hydrophobic material, such as a
hydrophobic PTFE membrane, preferably having a thickness of around
0.25 mm and a porosity of 0.2 microns. However, other suitable
forms of valves means will also be apparent to those skilled in the
art. The growth surface of the culture chamber or culture bag may
be treated to further enhance cell adhesion, for example by
charging the surface by bombardment with electrons. It is also
possible to modify the free --OH groups of the silicone rubber
surface to encourage attachment of various chemical moieties, in
the manner already described in relation to the invention in its
third aspect. Alternatively, cell attachment to the growth surface
of the culture chamber may be promoted by adapting the size of the
micro-cupules or depressions to the specific requirements of the
cells to be cultured. In a preferred embodiment, the culture
chamber further comprises a second chamber separated from the first
chamber by means of a semi-permeable membrane. The second chamber
preferably has an access means separate from that of the first
chamber.
In a fifth aspect of the invention, there is provided an apparatus
comprising a plurality of culture chambers according to the
invention in its fourth aspect, for use in a method of culturing
microbiological material. In an embodiment, the inlets of the
culture chambers are interconnected and the outlets of the culture
chambers are interconnected. In a preferred embodiment, the
apparatus has at least one further chamber(s) having a
semi-permeable wall that is positioned within each culture chamber,
each semi-permeable chamber(s) having an inlet that is
interconnected with the inlet of any other semi-permeable chambers
and having an outlet that is interconnected with the outlet of any
other semi-permeable chambers. In a preferred application,
anchorage-dependent stromal cells are grown on the textured surface
of the culture chamber(s), and anchorage-independent stem cells are
then inoculated into the culture chamber(s), to allow proliferation
of the stem cells. Preferably, the apparatus is a bio-reactor. The
bio-reactor is particularly applicable to the bio-processing of
liquors containing particular matter, such as blood cells or cell
debris. Conventionally, bio-reactors are normally closed systems
and, as such, have the disadvantages of relatively low productivity
and efficiency. One particular drawback is the limited volume of
oxygen available for reaction in such closed systems. Moreover,
such systems not normally suitable for the processing of liquors
containing particular matter, such as whole blood. The bio-reactor
according to the invention does not suffer from the aforementioned
problems because it comprises oxygen permeable walls, and a
textured surface of silicone rubber to assist the growth process of
the bio-substances. Thus, the desired product may be subsequently
generated in a continuous process by a passage of liquid nutrient
medium over the bio-substances. In a preferred embodiment, a method
of carrying out a bio-processing operation in a culture chamber or
an apparatus comprises attaching cells for performing the
bio-processing function to the textured surface of the culture
chamber(s), introducing liquor to be processed into the culture
chamber(s) via an inlet and collecting the processed liquor at an
outlet from the culture chamber(s). Preferably, the spent medium
including cellular by-products is removed from the culture
chamber(s), and fresh nutrient medium is passed through a
semi-permeable chamber(s) located within the culture chamber(s), so
to allow fresh medium to diffuse through the semi-permeable
membrane into the culture chamber(s). Preferably, the nutrient
medium is passed through the semi-permeable chamber in the opposite
direction to that in which the liquor or spent medium is passed
through the culture chamber. This has the advantage that cells
growing in those areas of the culture chamber having the most
heavily depleted medium are contacted with fresh medium first. The
nutrient medium may be recycled. In a preferred embodiment, the
apparatus is filled with liquid medium, which has first been
inoculated with a desired cell line. The assembly of reactor tubes
may then be arranged to be rotated or agitated, for example, using
machinery such as that employed for conventional roller bottles.
Rotation may be continued until cell confluence is obtained, as
evidenced by the levelling of the rate of glucose uptake. The inner
surfaces of the reactor tubes are, therefore, extensively coated
with the cells at this stage. If appropriate, rotation may be
interrupted for replacement of the medium in the reactor. The
reactor tubes can then be removed from the rollers and connected to
a suitable media reservoir. A continuous stream of liquid nutrient
medium may be arranged to pass through the reactor envelopes, the
product being harvested at the outlet. During this procedure, it is
desirable to provide an airflow over the reactor, to assist
oxygenation. In a preferred embodiment, the apparatus is especially
adapted for bio-processing of liquors containing particulate
matter, such as blood cells or cell debris. The continuous flow
system according to the invention is especially applicable to the
processing of whole blood, for example, in an artificial extra
corporeal organ substituting or supporting the functions of the
human liver. Advantageously, the system obviates the need of
separating the particulate matter prior to processing and then
having to reunite the constituents. It is also envisaged that the
culture chambers and apparatus according to the invention may have
other medical applications, such as for expansion of other primary
cell types, or for use as an ex vivo model for drug metabolism if
colonised with hepatocytes and the like. In another embodiment, the
culture chambers further include semi-permeable chambers positioned
within themselves, such as, for example, semi-permeable chambers
made of cellulose acetate. These semi-permeable chambers are
arranged to be separately connected to common inlets and outlets at
their respective ends. In this embodiment, the bio-processing
operation involves the following procedures. First, the cells grown
to perform the bio-processing function are attached to the textured
surface of the culture chamber. The culture medium is then removed
from said culture chambers. Next, the nutrient medium is passed
through the semi-permeable chambers, introduced from a reservoir
through the inlet at one end of the semi-permeable chambers,
issuing at the outlet on the opposing end. If desired, the medium
may be recycled from the outlet, to return again to the inlet of
the chambers. The liquid to be processed, such as blood, for
example, is then arranged to flow through the culture chambers, the
textured interior surface of which are now coated with cells. The
liquor is introduced for this purpose at the inlet of the culture
chambers, formerly serving at the medium inlet, and issuing at the
outlet on the opposing end. The liquor is preferably passed through
the culture chambers in opposing direction to that of the nutrient
medium. During this procedure, nutrients from the medium pass
through the semi-permeable chambers, traversing the stream of
liquor, to feed the cells adhering to the coating of the culture
chambers. At the same time, the semi-permeable chambers also
perform the function of cleansing the liquor of waste materials,
such as ammonia or urea, etc. The treated liquor is finally
collected at the outlet of the culture chambers. The productivity
and efficiency of the growth process, especially in the case of
anchorage dependent cells, can be substantially enhanced using the
bio-reactors according to the invention, especially when compared
with conventional reaction vessels that do not utilise oxygen
permeable containers and, thus, cannot sustain cell growth process
in the manner permitted by the invention.
In a sixth aspect of the invention, there is provided a well for
use in a method of culturing microbiological material having at
least one wall defining the well, at least a portion of the wall
being gas-permeable, to enhance oxygen supply to the well, and at
least a portion of the interior surface of the wall being textured,
to increase surface area and to enhance cell adherence. In a
preferred embodiment, the gas-permeable portion of the wall and the
textured portion of the wall are positioned at or near to the base
of the well. In another embodiment, the gas-permeable portion of
the wall comprises a gas-permeable membrane, preferably formed of
silicone rubber. The membrane preferably has a textured surface
facing the interior of the well, to increase available surface area
and to facilitate cellular attachment. In a preferred embodiment,
the textured surface has crater-like depressions or micro-cupules
and is preferably a textured silicon rubber layer made by a method
according to the invention in its second aspect. In a further
embodiment, at least a portion of the interior surface of the wall
comprises porous silicone rubber in accordance with the invention
in its third aspect, preferably near the base of the well. In an
especially preferred embodiment, the porous silicone rubber is
provided with a textured silicone rubber layer which serves to form
the interior surface of the well. Such wells are particularly
useful in cellular assays which require the cells to remain
substantially trapped in the wells, during a succession of steps
involving washing or treatment with various reagents.
In a seventh aspect of the invention, there is provided a
microtitre plate having at least one well according to the
invention in its sixth aspect. The wells help to increase both the
quantity of cells that can be grown in a microtitre well of a given
size, as well as their metabolic activity. Preferably, the
microtitre plate has at least one well having a wall at least a
portion of which comprises a textured silicone rubber surface in
accordance with the invention in its second aspect. As microtitre
wells become increasingly minimized in size, the number of cells
that can be grown in each well, for example, for drug metabolism
studies, is also reduced because of the decrease in available
growth surface area. Moreover, those cells that can be grown are
also starved of oxygen due to the decrease in gassing surface to
volume ratio. The microtitre plate according to the invention helps
to eleviate this problem by firstly increasing the available
surface area with the textured surface and secondly allowing the
cells to be gassed from below the second membrane.
In an eighth aspect of the invention, there is provided an implant
device comprising a cell support structure having a coating with a
textured surface, to promote anchorage of the implant by cell
attachment and ingrowth by surrounding tissue upon implant.
Preferably, the textured surface has crater-like depressions or
micro-cupules. In an especially preferred embodiment, the coating
comprises textured silicone rubber, preferably manufactured
according to the invention in its second aspect. The implant
devices according to the invention may take many different forms,
such as, for example, a heart valve, a sternum implant, or a
reconstructed calf ligament. The textured surface on the implants
acts as an anchor for tissue in-growth. Thus, the textured implant
can help to prevent migration of larger implants or to promote a
secure bond, where the interface with the implant and the
surrounding tissue is critical. It has been found that the textured
surfaces according to the invention have the further advantage of
helping to reduce the formation of capsule-type scar tissue
following implantation.
In a ninth aspect of the invention, there is provided a substrate
for growth of skin grafts in vitro comprising a flexible membrane
having a textured surface. A major problem associated with the
growth of skin ex vivo is that, when it is grown on a rigid or
solid surface, the skin tends to be bristle and does not have the
opportunity to "learn" to be flexible. In addition, the
undersurface of the skin tends to be smooth and scar-like, which
makes it difficult for the skin graft to take. The flexible
membrane used in the inventive substrate helps to prevent the skin
graft from becoming brittle, whilst the textured surface increases
the surface area available for cell adhesion, promotes cell
adhesion and helps to gives the skin a rough surface, so as to
enhance "taking" of the graft on transplant. In an embodiment, the
flexible membrane is gas-permeable, preferably comprising a
material such as silicone rubber. In a preferred embodiment, the
textured surface has crater-like depressions or micro-cupules.
Preferably, the textured surface is formed of silicone rubber
manufactured according to the invention in its second aspect. The
textured surface not only provides a greater surface area for cell
growth, but also allows a degree of ingrowth into the silicone
rubber in small areas, so that upon removal from the growth
surface, the skin undersurface will be textured, assisting the
taking process of the graft. In addition, the high oxygen
permeability of the silicone rubber would assist in promoting the
metabolic activity of he growing graft.
In a tenth aspect of the invention, there is provided a tissue
support structure for use in a method of culturing tissue or
cellular agglomerates, which comprises a biocompatible material
having an internal system of pores, the pores promoting cell
attachment and anchorage and oxygen supply to the tissue.
Microparticles of organs grown ex vivo have many applications in
the drug development industry. However, conventional support
substrates for tissues or tissue fragments grown ex vivo are
severely limited as to the size of the tissue agglomerates that may
be grow. The need to provide oxygen and nutrients to the centre of
a three dimensional tissue mass has been widely recognised by those
skilled in the art and has been addressed in a number of different
ways, all of which involve complex and expensive support structures
having specific structural features for gas and nutrient supply.
The tissue support structures according to the invention have a
system of pores and channels within the porous structure that is
capable of mimicking a biological capillary system, delivering
oxygen directly to the centre of the tissue growing on the
structure. This allows much larger agglomerates to be formed while
avoiding necrosis and apoptosis. In a preferred embodiment, the
porous material is provided with small, fine bore tubes. In another
embodiment, the shape of the porous material may be adapted so as
to engineer the shape of the resultant tissue. In a preferred
embodiment, the porous material comprises porous silicone rubber,
preferably made according to the invention in its third aspect. In
an especially preferred embodiment, the tissue support structure
comprises porous silicone rubber having small, fine bore tubes. In
this case, the silicone rubber may be provided with a porous and
micro-capillary structure by using finely ground sacrificial filler
and long thin needles of crystalline sacrificial filler in
admixture in the process according to the invention in its third
aspect, the ground filler serving to provide the porous
superstructure and the needles of filler serving to provide the
micro-capillaries. However, artificial capillaries may also be
introduced into the porous silicone rubber by other methods, such
as trapping gases in the setting silicone rubber, mechanical
disruption, laser ablation, etc.
In an eleventh aspect of the invention, there is provided an
apparatus for culturing tissue or cellular agglomerates comprising
a tissue support structure according to the invention in its tenth
aspect and a gas-permeable membrane, to enhance oxygen supply to
the system of pores and channels within the porous material, and
therefore to the tissue. Preferably, the gas-permeable membrane is
attached to the porous material. In a preferred embodiment, the
gas-permeable membrane is a silicone rubber, preferably made in
accordance with the invention in its third aspect. Preferably, the
porous material is attached to the gas-permeable membrane using a
gas-permeable adhesive, such as a silicone rubber adhesive. In a
preferred embodiment the plurality of tissue support structures are
arranged in close proximity to one another, so as to allow fusion
between tissue or cell masses growing on each structure, to create
larger tissue or cellular agglomerates. It is envisaged that tissue
grown on this type of structure could reach macro-dimensions being
fed with oxygen via diffusion through solid threads attached to
tubes through which oxygen would be passed. Preferably, the support
structure is in the form of a pillar, the dimension of which are
approximately 0.25 mm.times.2 mm.
In a twelfth aspect of the invention, there is provided an
artificial implant formed from a material having an increased
system of pores, the pores promoting cell attachment and anchorage
and oxygen supply to the cells on the implant surface. The pores
throughout the structure allow a degree of ingrowth and anchorage
of the cells, as well as a pathway for supply of oxygen to the
cells on the surface. In a preferred embodiment, the porous
material comprises porous silicone rubber, preferably made
according to the third aspect of the invention. In a preferred
embodiment, the artificial implant is adapted for use as a
cartilage implant. In such applications, the porous silicone rubber
is carefully selected to contain the necessary degree of
biologically inert filler to give it the required degree of
elasticity and/or flexibility of the cartilage. Preferably, the
porous material is seeded in vitro with chondrocites, to form a
layer of cartilage over the implant. Such an implant may be used
for replacing eroded joints and the porous silicone structure may
be moulded to conform to the shape of the bone it is to protect.
Prior to the present invention, cartilage intended for such
purposes was grown in vitro in a flat single layer on culture
plates and was then placed over the eroded bone. The principle
disadvantage of such a method is that the cartilage so grown is in
a flat form and does not readily accommodate to the contours of the
bone to be protected. The implants according to the invention do
not suffer from such a disadvantage and, thus, may be used to
provide "spare parts" for surgery in the human body. In another
preferred embodiment, the porous material of the cartilage implant
could be moulded into the shape of a nasal bridge, or an ear. This
type of permanent synthetic bio-compatible implant offers both
support and a degree of permanent protection to the cartilage
structure.
In a thirteenth aspect of the invention, there is provided an
artificial implant according to the invention in its twelfth aspect
in the form of a vascular graft. Conventional vascular grafts often
suffer an adverse fate because their base material has incompatible
physical properties to those of the native tissue. In a preferred
embodiment, the vascular graft of the present invention comprises a
hollow tube made from porous material, preferably porous silicone
rubber. In an embodiment, the interior surface allows cell
adhesion, and preferably endothelial cells are grown on the
interior surface of the graft. In another embodiment, the exterior
surface also allows cell adhesion, and preferably smooth muscle
cells are grown on the exterior surface of the graft. In a
preferred embodiment, one or both surfaces of the graft are
additionally roughened to enhance cell attachment, preferably by
providing the graft with textured silicone rubber surface. The
elastic, compression and oxygen transport characteristics of porous
silicone rubber closely mimic those of living tissue and, thus,
help permit common problems, such as stenosis, to be overcome. A
further advantage associated with the vascular grafts according to
the invention are that these produce a lamina flow and not the
turbulent flow associated with rigid synthetic grafts, hence
helping to minimizing the problems of thrombosis. Due to the
chemical properties of silicone rubbers, such vascular grafts would
also be resealable, which would be advantageous for patients
requiring repeated vascular access, for example, patients suffering
from renal disease and undergoing long-term kidney dialysis.
In a fourteenth aspect of the invention, there is provided a cell
implant means comprising a porous material for retention of cells
to be implanted, the pores promoting cell attachment and anchorage
and oxygen supply to the cells, and a protective means to shield
the cells from immune attack after implant. In an embodiment, the
porous material comprises silicone rubber, preferably made by the
method according to the invention in its third aspect. In a
preferred embodiment, the protective means desirably comprises a
semi-permeable membrane forming an envelope around the porous
material. In an especially preferred embodiment, the cell implant
means is adapted for use as an endocrine implant. The porous
material is seeded in vitro with endocrine cells, such as islets of
Langerhans cells. The development of fully-functional endocrine
implants, especially of the islets of Langerhans insulin-secreting
cells, has long been a target for clinical research. However,
expanding islet cells has proved extremely challenging, because it
is difficult to make them proliferate in culture. Islet cells from
foetal tissue have been proliferated in culture but permanently
lose function over time. The endocrine implants according to the
present invention should help to overcome such difficulties. On
implantation of the endocrine implant, the required hormone is
released through the semi-permeable membrane, whilst this membrane
also acts as a barrier to the body's own defences against the
foreign cells. Regulation of the hormones released can occur
naturally, as feedback control molecules are able to pass through
the semi-permeable membrane and communicate with the endocrine
cells directly.
In a fifteenth aspect of the invention, there is provided a drug
delivery system comprising a porous material whose pores have been
impregnated or saturated with a drug for delivery. Preferably, the
drug delivery is suitable for implantation into a human or animal
body. In a preferred embodiment, the drug is present in admixture
with at least one sustained release ingredient. In an especially
preferred embodiment, the porous material comprises a porous
silicone rubber, preferably made by a method according to the
invention in its third aspect. Many drugs exist as small molecules
which are capable of diffusing readily through silicone rubber.
Such drugs can be incorporated into the porous silicone rubber,
which acts as a drug delivery system. Advantageously, the porous
nature of the silicone rubber means that it is capable of exposing
a large surface area to bodily fluids for a relatively small
implant. Moreover, the synthetic nature of the silicone rubber
means that the system is less likely to be rejected by the body
when implanted. In addition, the material will not biodegrade as
with many of the current devices, making it possible to explant the
spent device for analysis and monitoring purposes if required.
In a sixteenth aspect of the invention, there is provided a
filtration media comprising porous silicone rubber, for use in
separations. Preferably, the porous silicone rubber is made
according to the invention in its third aspect. In a preferred
embodiment, the pores of the silicone rubber are of sub-micron
size, preferably in the order of 1 nm-10 .mu.m, more preferably in
the order of 10 nm-5 .mu.m, and more preferably about 0.1-0.5
.mu.m. The filtration media may be used in magnetic separation and,
in such a case, the porous silicone rubber preferably includes
magnetic additives. The filtration media may also be used in
expanded bed adsorption and, for this application, the porous
silicone rubber is preferably in particulate form. The filtration
media may be for use in static inline filtration, for which the
porous silicone rubber is preferably in the form of sheets or
tubes. In a preferred embodiment, the filtration media comprises
porous silicone rubber in the form or annular discs. Preferably,
porous silicone rubber with a sub-micron pore size is used. In
particulate form, the filtration media according to the invention
is highly suited for use in the burgeoning market of expanded bed
absorption technology. This is because porous silicone rubber can
be easily modified to have the appropriate density and, due to its
elastic nature, can be used in whole broth or continuous processes
over protracted periods of time. After primary processing, the
filtration media can be made receptive to all common moieties used
in affinity chromatography processes. In addition, the filtration
media according to the invention can also easily be made magnetic,
so that it can be easily separated from a whole broth system using
magnetic separation.
In a seventeenth aspect of the invention, there is provided a cell
cryopreservation system comprising a porous material for absorbing
cell culture into the internal system of pores and a container
suitable for storage in liquid nitrogen. In a preferred embodiment,
the porous material comprises porous silicone rubber, preferably
porous silicone rubber made in accordance with the invention in its
third aspect. The container preferably comprises releasable sealing
means. In an alternative embodiment, the container is a
syringe-type plunger. In such an embodiment, a number of
cylindrical particles of porous silicone rubber may be placed in a
tube fitted with a syringe type plunger. An operator could then
suck up the required culture to saturate the porous silicone rubber
particles and then store the device in liquid nitrogen. Upon
retrieval, the operator then has a number of porous silicone rubber
particles containing the same culture that can be used for several
inoculums.
In an eighteenth aspect of the invention, there is provided an
electrode comprising a porous material having electrically
conductive particles dispersed therein. In a preferred embodiment,
the porous material comprises porous silicone rubber, preferably a
porous silicone rubber made in accordance with the invention in its
third aspect. Preferably, the conductive particles are metal or
carbon powders. In another embodiment, the porosity of the
electrode material promotes adherence of microorganisms to the
electrode surface. In an especially preferred embodiment, the
microorganisms are capable of digesting waste, such that the
electrodes may be used in the treatment of sewage and in similar
applications. In another embodiment, the electrode forms part of an
electrode system comprising a plurality of electrodes immersed in a
liquid electrolyte and connected to an electric circuit. As in
conventional electrolytic systems, in use, two electrodes (a
cathode and an anode) are immersed in the liquid electrolyte, are
connected to an electric circuit with a potential being applied
between them. In special applications, electrolytic baths may
comprise a plurality of electrodes. The porous silicone rubber
electrodes have a number of advantageous features, including a
large surface area and, hence, a high electrical capacity,
robustness, inertness and resilience (aided by some degree of
elasticity). Such characteristics are particularly important in the
relatively hostile chemical and physical environment of agitated
liquid electrolytic cells. Furthermore, the porous silicone rubber
also provides a favourable surface for the growth of
micro-organisms making such electrodes particularly suitable for
special uses in water purification and sewage treatment
applications. Traditionally, such water treatments normally
comprise the functions of converting (a) carbonaceous material to
carbon dioxide and water, (b) nitrites to nitrates, and (c)
nitrates to atmospheric nitrogen, all three functions relying on
the actions of micro-organisms. Among difficulties associated with
conventional procedures are those of providing an adequate stream
of oxygen through the sewage to maintain the micro-organism
activity. This usually requires agitation of the liquid using
mechanical stirrers, while passing a stream of oxygen through the
sludge in the case of functions (a) and (b) and providing a safe
reducing atmosphere for (c). Using porous silicone rubber
electrodes according to the invention in, for example sewage
treatment, advantages are achieved in respect of greater output
efficiency, enhanced reliability owing to the absence of moving
parts in the system, as well as lower operating costs. By using the
porous silicone rubber electrodes, an oxygen stream is applied at
the anode to pass through the sewage, allowing micro-organisms to
effect the reactions (a) and (b), while an enhanced level of
hydrogen at the cathode aids the conversion by micro-organisms of
(c).
In a nineteenth aspect of the invention, there is provided a wound
dressing comprising a first layer of a porous gel and a second
layer of a carrier gel. In a preferred embodiment, the porous gel
layer comprises a porous silicone rubber gel, preferably made by a
method according to the invention in its third aspect. The carrier
gel layer may also comprise a silicone rubber gel. Preferably, the
carrier gel is applied to a supportive structure, such as a
Dacron.RTM. mesh. In a preferred embodiment, the porous gel layer
is infused with a drug for delivery to the wound, such as a
growth-promoting drug or an antibiotic. The silicone rubber wound
dressing according to the invention also helps to control scar
formation by leaching low molecular weight silicones into the
wound, a technology already employed in the field. The silicone
rubber wound dressing has the added advantage of increasing the
contact area with fluids from the wound, thereby improving leaching
and allowing greater oxygen transport to the site, whilst
maintaining asepsis. Moreover, the dressing permits drugs infused
into the porous structure to leach into the wound over a prolonged
period of time to aid the healing process.
In a twenteeth aspect of the invention, there is provided a
clinical swab comprising a porous material, the pores increasing
the surface area of the swab and promoting oxygen transport to the
swab surface. In a preferred embodiment, the porous material
comprises porous silicone rubber, preferably made by a method
according to the invention in its third aspect. In a further
embodiment, the porous material contains a radio-opaque additive,
such as barium sulphate. This allows any lost swabs to be easily
traced and then removed. In another embodiment, the porous material
is infused with a drug. The swab is preferably attached to the end
of a stick made, for example, of wood or plastic. The swab
according to the invention has a number of advantages over the
conventional swabs. The porous silicone rubber is oxygen permeable.
The silicone rubber is also non-limiting, reducing the risk of
debris being left behind after use. The silicone rubber is also
better attached to the stick than cotton wool in conventional
swabs. The silicone is furthermore chemically very stable and will
also allow microorganisms to adhere to the swab surface.
BRIEF DESCRIPTION OF THE DRAWINGS
In order that the invention may be better understood, examples of
the various aspects will now be described, by way of illustration
only and with reference to the accompanying drawings, wherein:
FIGS. 1, 2 and 3 show successive steps of the manufacturing process
in accordance with the second aspect of the invention;
FIG. 4 shows a cross-sectional view of three-dimensional porous
silicone rubber in accordance with the third aspect of the
invention;
FIG. 5 is a schematic plan view of a culture bag in accordance with
the fourth aspect of the invention;
FIG. 6 is a schematic cross-sectional view of the culture bag of
FIG. 5;
FIG. 7 is a cross-sectional view of a membrane wall of the culture
bag of FIGS. 5 and 6;
FIG. 8 is an exploded view of the valve of the culture bag of FIGS.
5 and 7;
FIG. 9 is a diagrammatic illustration of a bio-reactor apparatus
according to the fifth aspect of the invention;
FIG. 10 is a silicone rubber tube from the bio-reactor of FIG.
9;
FIG. 11 is a cross-sectional side view of a bio-reactor apparatus
with dialysis tubes;
FIG. 12 shows plan view of a microtitre plate according to the
sixth aspect of the invention;
FIG. 13 shows a cross-sectional view of the plate of FIG. 12 along
line A-A';
FIG. 14 shows a schematic artificial capillary system, in
accordance with the ninth aspect of the invention; and
FIG. 15 shows a part cross-sectional view of an endocrine implant
according to the thirteenth aspect of the invention.
DETAILED DESCRIPTION OF THE INVENTION
FIGS. 1, 2 and 3 show successive steps of the manufacturing process
in accordance with the second aspect of the invention. In FIG. 1,
the surface of a substrate 10 is coated with a layer of uncured
silicone rubber precursor 11. In FIG. 2, a sacrificial filler 12,
such as sodium chloride, is applied to the silicone rubber layer 11
whilst the latter is still tacky, the sodium chloride 12 becoming
adhered to and partially embedded in the silicone rubber layer. Any
excess sodium chloride 12 that is not adhered to the silicone
rubber layer 11 is removed and the silicone rubber layer 12 is
allowed to cure. Once the silicone rubber layer 11 has been cured,
the sodium chloride 12 is dissolved in a solvent, such as water,
leaving craters or micro-cupules 13 forming a textured surface
structure 14 as shown in FIG. 3.
In FIG. 4, a porous silicone rubber article 70 has a textured
exterior surface with craters 71 and pores 72 within the body of
the silicone rubber article 70, forming porous channels throughout
the three dimensional structure. The porous silicone rubber article
70 is made from GE Silicone's LIM 6070-D2 (part A & B) or
McGhan NuSil's MED 4970 (part A & B), to form the silicone
rubber and J. Astley & Sons Food Grade NaHCO.sub.3 (sodium
bicarbonate) as sacrificial filler. Stainless steel powder (MBC
Metal Powders Ltd 316L SS fines 325 mesh) is also added for a high
density silicone rubber product. The sodium bicarbonate is mixed
with each of parts A and B of the silicone rubber separately, at a
ratio of 3:1 w/w. The mixing is carried out using a conventional
Z-blade mixer, although other mixer types may be used, or mixing
may even be performed by hand. Stainless steel powder is added to a
level to give the desired density (although other high mass
powders, such as titanium oxide, can also be used). Once mixed with
the sodium bicarbonate, parts A & B are stored separately in a
cool place for further processing. The components must be kept
apart as one contains the accelerator and the other the catalyst
that will cause curing. If cross-contamination of the parts occurs,
the material will start to cure. When ready to cure the material
into the required shape, parts A & B are mixed together on a
two roll mill for 15 to 20 minutes to ensure complete mixing. The
resultant mixture is then fed into a cold head extruder and
extruded through a die of the appropriate shape. The resultant
extrudate is picked up by a heat resistant conveyor and passed
through a hot box set to such a temperature that the extrudate
itself is heated to 175.degree. C. This facilitates the cure of the
material without allowing the sodium bicarbonate to decompose and
hence "blow" the material. Depending upon the geometry of the
extrudate, it is passed through either a rotary cutter (for small
cross-sections) or a reciprocating cutter (for larger geometries)
and chopped into the appropriate particulate shape. This "preform"
is the stored in a dry place until further processing is required.
When required, the material is boiled in at least a five-times
excess of pyrogen-free water for one hour. This process is repeated
four or five times or until no further traces of sodium bicarbonate
are evident, as indicated by the pH of the water. The material is
then finally rinsed in pyrogen-free water, bottled in an excess of
the same and autoclaved to facilitate sterile storage. The material
is now in a form ready for sale as a stand alone support
matrix.
In a further example, the porous silicone rubber article 70 is made
GE Silicone's RTV (room temperature vulcanising) 615 (part A &
B), as the silicone rubber material, and J. Astley & Sons Food
Grade NaHCO.sub.3 (sodium bicarbonate), as the sacrificial filler.
For a high density silicone product, iron oxide (magnetic
precipitate) from Fishers Scientific Products is used. The sodium
bicarbonate is wet milled under xylene using a Biaton bead mill to
a particle size of 0.1 to 0.4 .mu.m. This range can be further
narrowed by separation in a Malvern.RTM. particle sizer. Using
these methods, a whole range of particle sizes and distributions
can be achieved. The sodium bicarbonate is mixed with each of parts
A and B of the silicone rubber separately, at a ratio of 3:1 w/w.
The mixing is carried out using a conventional Z-blade mixer,
although other mixer types may be used, or mixing may even be
performed by hand. If the density is to be increased, the iron
oxide is added to a level to give the desired density. Other high
mass powders such as titanium oxide can also be used. Further
weighting or magnetic moieties may also be mixed in, if required.
Once mixed with the sodium bicarbonate, the parts A & B are
stored separately in a cool place for further processing. When
ready to cure the material into the required shape, parts A & B
are mixed together on a two roll mill for 15 to 20 minutes to
ensure complete mixing. Again other apparatus could be used. The
resultant mixture is then fed into a cold head extruder and
extruded through the open scroll and collected as ingots on trays.
The ingots are then cured at 150.degree. C. in a standard
convection oven. The ingots are then ground in a mill to the
required size and can again be sized using a Malvern.RTM. particle
sizer if required. This "preform" is the stored in a dry place
until further processing is required. When required, the material
is boiled in at least a five-times excess of pyrogen-free water for
one hour. This process is repeated four or five times or until no
further traces of sodium bicarbonate are evident, as indicated by
the pH of the water. The material is then finally rinsed in
pyrogen-free water, bottled in an excess of the same and autoclaved
to facilitate sterile storage. This product is biocompatible, it
has pores in a very well defined size range and of an amorphous
geometry.
In FIGS. 5 and 6, a culture bag 20 comprises two membranes 28
joined at their outer edges 27, each membrane 28 having a textured
(interior) surface 26. Inlet and outlet ports 23 extend between the
inside and the outside of the bag, each port 23 being provided with
a valve 24. A degassing valve 22 is provided in the centre of one
of the membranes 28, this membrane 28 being uppermost when the bag
20 is in use. In FIG. 7, each bag membrane 28 is prepared by
covering the edges 27 of a smooth silicone rubber sheet 25 with a
mask (not shown) and applying a layer of room-temperature
vulcanising liquid silicone rubber to the exposed central portion
of the sheet. Next, vacuum-dried salt is sprinkled over the layer
of liquid silicone rubber so that it is uniformly covered. The
liquid silicone rubber is then cured and the salt is washed out,
producing a membrane 28 with a cratered or micro-cupulated surface
26. In FIG. 8, a degassing valve is formed by first cutting a hole
31 out of the centre of one of the membranes 28, over which the
valve will be placed. A washer 29 made of uncured silicone rubber
is positioned around the hole 31 on the smooth (outer) face of the
membrane 28. A hydrophobic PTFE membrane 30 with 0.2 .mu.m pores
and a thickness of 0.25 mm is laid over the washer 29, and a second
washer 29 is placed on top. This is then repeated with a second
PTFE membrane 30 and a third washer 29. When the bag 20 is to be
assembled, the two silicone rubber membranes 28 are laid on top of
one another, with the rough surfaces together. Two lengths of
tubing for the inlet and outlet ports 23 are placed between the
silicone rubber membranes 28, protruding slightly into the rough
area. The ports 23 are provided with valves 24. Next, room
temperature vulcanising silicone rubber is applied to the
untreated, smooth edges 27 of the silicone membranes 28, along
which the membranes 28 are to be joined to form a bag configuration
20. Uncured silicone rubber is applied around the tubing where it
lies adjacent to the smooth edges of the membranes 28. The
constituents of the culture bag 20 so arranged are then welded or
glued together using elevated temperatures and pressure. The edges
of the silicone membranes 28 are sealed to form a bag 20, the
degassing valve is formed from the layers of washers 29 and PTFE
membrane 30, and the tubing for the ports 23 becomes integrated
into the bag structure 20.
In FIG. 9, a bio-reactor apparatus comprises two reactor tubes 40
(in practice, a larger number, such as seven or eight tubes, is
generally preferred). Each reactor tube 40 carries an internal
coating of textured silicone rubber 41. In use, in order to grow
the cells on interior surfaces 41 of tubes 40, medium carrying cell
lines is introduced through an inlet 43. Reactor tubes 40 are
interconnected through distributors 42. One or more strengthening
members 45 ensure rigidity of the assembly. The assembly is rotated
on rollers (not shown), followed by evacuation of the liquid and
subsequent passage of nutrient medium over the cells. The medium is
introduced through the inlet 43 and issues from the outlet 44. The
product is finally collected at the outlet 44. In FIG. 10, the
reactor comprises a non-porous silicone rubber tube 40 carrying an
internal coating of textured silicone rubber 41. In FIG. 11,
dialysis tubes 51 are co-axially positioned within the reactor
tubes 40. Cells are grown in the annular space 52 by the passage
via introduction of medium comprising the cell line through the
inlet 47. After removal of the liquid from the annular space
through the outlet 48, the nutrient medium is passed through the
dialysis tubes 51 via medium inlet 49, issuing at outlet 50. At the
same time, the liquor to undergo the bio-reaction is passed through
the reactor tubes via inlet 47, for collection at outlet 48.
In FIGS. 12 and 13, a standard microtitre plate 60 has wells 61
without base walls (either conventional microtitre plates are used
and the base walls of the wells removed, or a microtitre plate is
produced without any base walls). A non-porous silicone membrane 62
is attached to the bottom of the wells, the membrane comprises a
silicone rubber sheet 63 having a coating with a textured surface
64 facing the area defined by the wells.
In FIG. 14, a tissue growth support structure comprises a tissue
mass 83, such as HT-29 (intestinal carcinoma) cells grown on
pillars of porous silicone rubber 81, the pores acting as a
capillary system, supplying oxygen to the cells in the centre of
the tissue mass 83. The pillars 81 are attached to a gassing
membrane 80 in a bio-reactor configuration, using gas permeable
silicone rubber adhesive 82. The oxygen diffuses through the
gassing membrane 80 and through the system of pores and channels to
reach the tissue agglomerate 83. The tissue growth support
structure permits much higher densities of HT-29 cells than
conventional systems.
In FIG. 15, islet of Langerhans cells are immobilized within a
bio-wafer 90 consisting of a disc of porous silicone rubber 92, in
which the islet cells are attached, sandwiched between
semi-permeable membrane layers 91, which allow insulin out but stop
host immune cells from attacking and destroying the transplanted
islet cells.
* * * * *